Project 1 : AdV endosome membrane penetration and cellular response

Summary : Adenoviruses are large, non-enveloped DNA viruses with a capsid diameter of 90nm. The capsid shields the 36kbp liner dsDNA viral genome (vDNA) on the way from the cell surface to the nuclear pore complex. To reach the cytosol AdVs penetrate the endosome after uptake into cells.
In this project we study the pertinent question; How do AdV and cell interact during viral endosome penetration? We focus on i) the mechanistic aspects of membrane penetration, ii) how the cell mounts autophagy/inflammation in responses to the virus endosome penetration and iii) how the virus in return controls the cellular response.

Figure 1: Endosomal escape of an individual adenovirus particle (green signal) from a ruptured endosome (red signal) in approximately real time. Virus particles are labelled with Alexa488, ruptured endosomes with mCherry-Galectin3 (published in Martinez et al. PMID 23996260, Maier et al. PMID 22855481).

Project 2 : Early fate of nuclear AdV genomes and the onset of replication

Figure 2 : Dynamic modification of adenoviral chromatin during the viral life cycle. Incoming genomes are densely packed inside the capsid into viral chromatin through the DNA binding protein VII. Imported viral genomes are decondensed, transcriptionally activated, replicated, recondensed and packaged into progeny virus. To adopt such diverse functional states viral genomes undergo dynamic chromatin remodelling using viral and cellular factors.

Summary : Most DNA viruses use the nuclear environment to replicate their genomes. The nuclear envelope (NE) is a major barrier on the way into the nucleus. This requires strategies for genome delivery as DNA nuclear import is not a physiological process. Inside the nucleus the compacted “transport” form of the vDNA has to reach the site of transcription/replication by tethering the vDNA to specific host chromatin. Transcription/replication than requires conversion of the vDNA into open chromatin while retaining the need to prevent vDNA from cellular sensors and antiviral effectors.
In this project we ask; What happens to viral genomes after nuclear delivery and prior to replication? We use adenoviruses (AdV) and more recently polyoma viruses (BKV) and ask how they initially i) release their vDNA from the protective capsid, ii) transport the released vDNA to the site of transcription/replication, iii) tether the vDNA inside the nucleus and engage in chromatin reorganization to start viral gene expression/replication, iv) decompact the viral genome from a highly condensed transport form into a transcription/replication template and v) prevent antiviral sensors and effector from vDNA inactivation.

Project 3 : Spatial organization of the nucleus during the AdV replication cycle

Summary: The short time window of few hours between the arrival of the genome in the nucleus and the onset of its transcription/replication is poorly understood. Its characterization will help identify host factors such as histone chaperones that target invading genomes and characterize their importance in transcription/replication. Morphologically distinct nuclear replication compartments form and replicated genomes are chromatinized and transcribed and subsequently condensed and packaged into progeny. In this project we want to understand: What drives nuclear compartmentalization during viral replication and (reversible) genome chromatinization? We use adenoviruses (AdV) and to some extend polyoma viruses (BKV) to investigate i) when and where replication compartments form, ii) what role does liquid-liquid-phase-separation (LLPS) has in creating replication compartments iii) how does replication change the biological properties of the replication compartment and where do replicated genomes accumulate/are packaged into progeny and iv) can we develop assays to screen compounds as inhibitors and/or modulators of virally induced LLPS to target viral replication/packaging.

Figure 3: In vivo viral genome replication (green signal) and replication centre formation (magenta signal) during the adenovirus life cycle at 1 frame/20min. Viral genomes are labelled with the AnchOR3 system and replication centres with USP7-mCherry (published in Komatsu et al. PMID 29997215).

Project 4 (emerging topic) : Replication of SARS-CoV-2 in differentiated lung epithelia

The SARS-CoV-2 coronavirus is the etiologic agent causing a worldwide pandemic with over 150.000 confirmed cases and nearly 30.000 resulting deaths in 2020 in France alone. In response to an ANR Flash Covid-19 call our group is partner in two selected projects “ANACONDA” directed by Marie-Line Andreola and “VasCOV” directed by Andreas Bikfalvi. ANACONDA started in April 2020 and uses fully differentiated airway epithelia derived from biopsy samples from individuals pertinent to different risk factors (age, gender, COPD). We investigate parameters of infection following SARS-CoV-2 exposure to correlate infectivity and immunity. In the VasCOV project we investigate the impact of SARS-CoV-2 on blood vessels and the Ace system using a unique 3D model. Our part in both project is the direct detection of viral RNA and viral proteins using imaging techniques such as IF and RNAscope to determine which cells are the primary and/or secondary targets of SARS-CoV-2 and to visualize morphological changes. Furthermore we use the SARS-CoV-2 model to develop in vivo RNA imaging to expand our expertise in the life cell imaging of viral nucleic acids and host-pathogen interactions (see project 2&3).